Rotaviruses have a double stranded segmented RNA genome and hence undergo genetic reassortment at high frequency. We have produced a series of temperature sensitive mutants of both UK (bovine) and Rhesus rotavirus. We have used these ts mutants to isolate reassortant rotaviruses from the growth yield of cells coinfected with a variety of human rotaviruses and ts bovine rotavirus. These reassortants were used to ascertain the gene coding assignments for human rotavirus neutralization specificity (gene 8 or 9), subgroup specificity (gene 6) and growth restriction in tissue culture (gene 4). In addition the human-bovine rotavirus reassortants were used to define the serotypic diversity of human rotaviruses (four or more serotypes). In addition, we isolated a series of reassortants derived from the growth yield of cells coinfected with ts UK and ts rhesus or ts UK, and canine rotaviruses. We characterized the phenotype of these reassortante and showed that: (1) neutralization specificity and hemagglutination reassort independently and hence are coded for by different genes; and (2) protease enhanced viral growth co-segregates with viral hemagglutination and hence these properties are coded for by the same gene. An understanding of the specific functions of the genes of rotavirus may have important implications for vaccine development. In addition, reassortant viruses represent potential vaccine virus candidates.